The objectives of the proposal are to isolate, culture and identify tracheal epithelial cells and to study their normal and neoplastic differentiation in monolayer, and upon combining with connective tissues. At present a suitable system for this purpose is not available. Cell isolation techniques will aim to obtain epithelial cells from rodent trachea using enzymes that selectively eliminate elastic tissue, collagen and fibroblasts. Elimination of fibroblast contamination from epithelial cell preparations will be attempted by using the differential adhesiveness of these two cell types and by treatment with collagenase of cells in a monolayer. A culture medium selectively promoting proliferation of epithelial cells will be devised. The role of hormones, vitamins and D-amino acids will be tested in this regard. Epithelial cells will be characterized in culture by cell and colony morphology and histochemical staining. Growth characteristics, cell cycle phases, in vitro life span and establishment of epithelial cell lines will be studied. Attempts will be made to induce differentiation in monolayer by providing vitamin, hormones and cyclic AMP. Epithelial identity will be tested in vivo by inoculating cultured cells inside x-irradiated tracheal grafts bearing a basement membrane where the epithelial cells are expected to undergo normal differentiation. Epithelial identity will be tested in vitro by the capacity to form basement membrane in the presence of mesenchymal cells. Neoplastic potential will be examined after treatment of epithelial cells with chemical carcinogens and sarcoma or other viruses, and subsequent inoculation inside x-irradiated grafts. Normal tracheal grafts will be tested for value in studies of carcinogenesis and co-carcinogenesis.